Investigating Tumorigenic Role Of ARHGEF10L In Hepatocellular Carcinoma

BackgroundIn the worldwide,liver cancer is one of the most common malignant tumors and hepatocellular carcinoma(HCC)accounts for more than 90%among all liver cancer.In our country,liver cancer ranks the second most common cancer.Various pathological factors can induce the occurrence and development of hepatocellular carcinoma.Hepatocellular carcinoma is considered to be the result of the interaction between different environmental risk factors and genetic factors.A variety of genetic factors and their epigenetic regulation are closely related to the occurrence of hepatocellular carcinoma,including the inactivation of tumor suppressor p53 protein,abnormal activation of Wnt/wingless signaling pathway caused by mutations of?-catenin and Ras/Raf/MEK/ERK and PI3K/Akt/mTor protein,abnormal activation of ErbB receptor protein family and mesenchyme epithelial transformation factor(MET)receptor proteins.The Ras homologous(Rho)small GTPase family containing about 20 human members,represents a subfamily of Ras superfamily of small GTPases(20-30 kDa),of which the most extensively characterized are Racl,RhoA and Cdc42 proteins.The activity of Rho GTPases is mainly regulated by three kinds of proteins:guanine nucleotide exchange factors(RhoGEFs),GTPase-activating proteins(RhoGAPs)and guanine nucleotide dissociation inhibitors(GDIs).Aberrant activity of Rho small G-proteins and their regulators are hallmarks of various cancers.And in hepatocellular carcinoma,the abnormal expression of GTP-RhoA can directly affect the process of proliferation,invasion and metastasis of tumor cells.Human ROCK(Rho-associated coiled-coil containing protein kinase)is one of major downstream effectors of small GTPase RhoA.After RhoGEFs activates the GTP-RhoA.the activated GTP-RhoA interacts with ROCK protein.ROCK protein family plays the central role in the organization of the actin cytoskeleton and is involved in a wide range of fundamental cellular functions such as contraction,adhesion,migration,proliferation and apoptosis.The activated ROCK protein further activates the downstream protein ezrin-radixin-moesin(ERM)and increases its phospho-ERM(pERM)levels.Rho guanine nucleotide exchange factor 10-Likel(ARHGEF10L),a member of the RhoGEF family(guanine nucleotide exchanging factors),contains the typical Dbl homology domain,a putative WD40-like domain and two predicted transmembrane helices.ARHGEF10L can induce GDP/GTP exchange at RhoA,but not at Racl or Cdc42.In addition,the SNP rs2256787 and rs10788679 of ARHGEF10L gene were associated with epithelial ovarian cancer(EOC)risk.The above studies suggest that ARHGEF10L,as a member of GEFs,played an important role in tumorigenesis.Epithelial-mesenchymal transition(EMT)is the transient and reversible change from a polarized and epithelioid to a fibroid and mesenchymal cellular phenotype,showing high migration and invasiveness.Studies have shown that EMT plays the important role in the process of cell proliferation,invasion and metastasis im various cancers including hepatocellular carcinoma,breast cancer and lung cancer.Further exploration of the relationship of ARHGEF10L and EMT has important clinical significance for the pathogenesis of hepatocellular carcinoma.In this study,we investigated the possible association between the candidate SNPs in the ARHGEF10L locus and various tumors,especially liver cancer.By comparing the results of the Illumina 384-SNP VeraCode microarray,Sequenom MassARRAY and Taqman genotyping assays,we determined which tumor risk(s)has genetic association with ARHGEF10L encoding gene.Based on the above genetic analysis,we investigated ARHGEF10L expression in hepatocellular carcinoma tissues using western blot and immunohistochemistry.We observed effects of ARHGEF10L expression on cell proliferation and cell migration.Then,BALB/c nude mice were used to generate the tumor-bearing animal model to verify the effect of ARHGEF10L expression on tumor growth.We determined the possible molecular mechanism of ARHGEF10L in carcinogenesis process of hepatocellular carcinoma by detecting the related molecules of RhoA signaling pathway and EMT.The purpose of this study is to explore the role and molecular mechanism of ARHGEF10L in the pathogenesis of hepatocellular carcinoma,and this provided a new theoretical and experimental basis for further treatment of hepatocellular carcinoma.Methods1.Illumina 384-SNP VeraCode microarrays,SequenomMassARRAYs and TaqMan genotyping were applied to investigate the possible correlations between tag single nucleotide polymorphisms(tag SNPs)of the ARHGEF10L gene and various tumor risks.SNPs rs2245148.rs35812446,rsl2732894,rs12756041,rs10788678,rs1193340,rs1193339,rs108880455 rs74059378,rs12117763,rs12562076,rs3766307 and rs35255680 were studied using Illumina 384-SNP VeraCode microarrays.SNPs rs10888045.,rs3753378.,rs2244444,rs12067869,rs78944230,rs12562076 and rs3766307 were studied by SequenomMassARRAYs.SNPs rs10888045,rs2244444,rs74059378 and rsl2732894 were investigated using TaqMan genotyping.2.Hepatocellular carcinoma tissue samples and tissues adjacent to the tumors(n=8)were collected at Shandong Provincial Qianfoshan Hospital.Using western blot,total protein was extracted to determine ARHGEF10L expression in hepatocellular carcinoma tumor tissues.All patients provided their signed informed consent.and the study was approved by the Ethics Committee at Shandong Provincial Qianfoshan Hospital(Approval number 2015005).Immunohistochemistry was performed to detect the ARHGEF10L expression in a panel of hepatocellular carcinoma tissue arrays(Alenabio,Xian,China).3.In order to detect the cellular biological function of ARHGEF10L in HCC,human hepatocellular carcinoma cells Be17402 and HepG2 were transfected with anti-ARHGEF10L siRNA and Allstars using the HiPerFect Transfection Reagent(QIAGEN,Germany).At the same time,Be17402 and HepG2 cells were transfected with an ARHGEF10L-overexpression plasmid and mock plasmid using PolyJetTM In Vitro DNA Transfection Reagent(SignaGen,USA).Western blot analysis was used to detect the ARHGEF10L expression level.In Be17402 and HepG2 cells,cell counting kit-8 assays(CCK-8)and clone formation assays were used to observe the effects of ARHGEF10L on cell proliferation,and transwell assays were used to determine the effects of ARHGEF10L on cell migration.4.In order to verify the effect of ARHGEF10L expression on tumor growth in tumor-bearing mice,BALB/c(Vital River,Beijing,China)nude mice were used to generate the tumor-bearing animal model.Bel7402 cells transfected with the lentivirus packing shARHGEF10L vector(GenePharma,Shanghai,China)were injected into nude mice to generate liver tumors.Magnetic resonance imaging(MR])studies was used to observe the changes of signal intensity in diffusion-weighted Imaging(DWI)and determine the changes of Apparent Diffusion Coefficient(ADC)value.At the same time,tumor tissues were taken out to measure the tumor volume and tumor cell structure was observed using Hematoxylin-Eosin staining(HE).5.In order to determine the effect of ARHGEF10L expression on RhoA signaling,this study used Activation Assay Biochem kit(Cytoskeleton,Denver,USA)to detect the activity of GTP/GDP-Rho GTPases.Then,in human hepatocellular carcinoma Be17402 and HepG2 cells,western blot investigated the influence of ARHGEF10L on ROCK1 and pERM protein expression.And pERM protein expression was detected in Be17402 and HepG2 cells transfected with anti-ROCK1 siRNA.6.Hepatocellular carcinoma tissue samples and tissues adjacent to the tumors(n=8)were collected at Shandong Provincial Qianfoshan Hospital.Using western blot,total protein was extracted to determine ROCK1 protein expression in hepatocellular carcinoma tumor tissues.7.In order to investigate the effect of ARHGEF10L expression on the induing of EMT,tumor tissue proteins of nude mice were extracted to detect changes of EMT-related markers(E-cadherin,N-cadherin,Vimentin,VEGF and Slug)by western blot.Then,in Be17402 and HepG2 cells with ARHGEF10L overexpression,changes in cell morphology were observed.Using western blot and immunofluorescence,these protein levels of EMT markers(E-cadherin,N-cadherin,vimentin and Slug)were clarified in Be17402 and HepG2 cells transfected with anti-ARHGEF1OL siRNA or ARHGEF10L-overexpressing.Results1.Illumina 384-SNP VeraCode microarrays found that SNP rs35812446,rsl2732894,rs74059378 and rs12562076 showed signifcant differences in allelic frequency and genotypic frequency between liver cancer and controls.Sequenom MassARRAYs found that SNP rs2244444 showed signifcant differences in allelic frequency and genotypic frequency between liver cancer and controls.TaqMan genotyping found that SNP rs2244444 and rs 12732894 showed signifcant differences in allelic frequency and genotypic frequency between liver cancer and controls.SNPs rs2244444 and rs12732894 in the ARHGEF10L-encoding gene is genetically susceptible to liver cancer risk.2.Compared to peritumoral tissue samples,hepatocellular carcinoma samples expressed more ARHGEF 10L expression using western blot and IHC.Therefore,ARHGEF 10L may be closely related to the pathogenesis of hepatocellular carcinoma.3.Using cell counting kit-8 assays(CCK8),clone formation assays and transwell assays,ARHGEF10L overexpression stimulated cell proliferation and migration in Be17402 and HepG2 cells.The inhibition of ARHGEF10L expression significantly retards cell proliferation and migration in Be17402 and HepG2 cells.These results suggest that ARHGEF10L could enhance the proliferation and migration of HCC cells in vitro.4.In xenograft tumor mouse model,the inhibition of ARHGEF 10L expression could suppress cell activity and tumor growth.Compared to the tumors from blank vector-transfected cells,tumors that originated from shARHGEF10L-transfected Be17402 cells showed decreased tumor volume,less DWI signal density and higher ADC value.Additionally,HE staining showed that tumors in mice injected with shARHGEF10L-transfected Be17402 cells exhibited lower cell densities.lacked compact tissue structures and showed many apoptotic-like cells.These results indicated that ARHGEF10L could promote cell proliferation in nude mice.5.In Be17402 and HepG2 cells,ARHGEF10L overexpression could promote the activity of GTP-RhoA and had no effects on GTP-RhoB and GTP-RhoC.Western blot detected increased ROCK1 and pERM protein expression in ARHGEF10L-plasmid transfected Be17402 and HepG2 cells.Conversely,Be17402 and HepG2 cells with anti-ARHGEF10L siRNA showed the decreased expression of ROCK1 and pERM protein.In Be17402 and HepG2 cells,inhibiton of ROCK 1 expression with anti-ROCK1 siRNA reduced expression of pERM protein.ROCK1 protein was higher expression in hepatocellular carcinoma samples than paratumor tissue samples.The results revealed that ARHGEF10L regulated the pathogenesis of hepatocellular carcinoma through the GTP-RhoA-ROCK1-pERM signaling pathway.6.Be17402 and HepG2 cells transfected with ARHGEF10L-expressing plasmids lost cell-cell contacts and exhibited a fibroblastic-like morphlolgy.In xenograft tumor mouse model,inhibition of ARHGEF10L expression decreased the levels of vimentin,N-cadherin,Slug and VEGF proteins.However,the E-cadherin protein did not change.Compared with cells transf-ected with mock plasmids.both Be17402 and HepG2 cells transfected with ARHGEF10L-expressing plasmids showed upregulated expression of mesenchymal markers(vimentin,N-cadherin,Slug),while the expression of epithelial marker(E-cadherin)was downregulated.Meanwhile,the protein level of E-cadherin were upregulated and the protein levels of vimentin,N-cadherin and Slug were downregulated in Be17402 and HepG2 cells with anti-ARHGEF10L siRNA.The above results indicated that ARHGEFIOL could promote the occurrence of EMT in hepatocellular carcinoma.Conclusions1.Illumina 384-SNP VeraCode microarrays,Sequenom MassARRAYs and TaqMan genotyping found that SNPs rs2244444 and rs 12732894 in the ARHGEF1OL gene were genetically susceptible to liver cancer risk.2.Significantly increased expression of ARHGEF10L protein was detected in hepatocellular carcinoma samples,proving that ARHGEF10L was strongly associated with hepatocellular carcinoma.3.Cell Counting Kit-8 assays,clonal formation assays and transwell assays proved that overexpression of ARHGEF110L promoted proliferation and migration of Be17402 and HepG2 cells.4,Inhibition of ARHGEF10L expression decreased tumor size and molecular activity in a xenograft tumor mouse model,revealing that ARHGEF10L expression plays an important role in the carcinogenesis of hepatocellular carcinoma.5.In Be17402 and HepG2 cells,overexpression of ARHGEF10L could stimulate the activity of GTP-RhoA and upregulate the level of ROCK1 and pERM protein.Inhibiton of ROCK1 expression reduced expression of pERM protein.Meanwhile,the expression level of ROCK1 protein in hepatocellular carcinoma tissues is significantly increased.These results indicate that ARHGEF10L could mediate the carcinogenesis of hepatocellular carcinoma through the GTP-RhoA-ROCK1-pERM signaling.6.ARHGEF10L overexpression downregulated the E-cadherin expression and upregulated the expression of vimentin,N-cadherin,Slug in Be17402 and HepG2 cells,revealing that ARHGEF10L could induce the occurrence of EMT in hepatocellular carcinoma and promoting the pathogenesis of hepatocellular carcinoma.To sum up,in hepatocellular carcinoma,expression of ARHGEF10L protein was increased and ARHGEF10L could contribute to the tumorigenesis of hepatocellular carcinoma through the GTP-RhoA-ROCK1-pERM signaling and the induction of EMT.